Effect of Lys175 mutation on structure function properties of Propionibacterium shermanii superoxide dismutase.
The role of electrostatic factors in the enzyme-substrate encounter process of manganese and iron containing superoxide dismutases has been studied in the enzyme from Propionibacterium shermanii by chemical neutralization of lysine residues and site-directed mutagenesis of the highly conserved residue Lys175. Lysine residues have been neutralized by carbamoylation and Lys175 has been selectively replaced by isoleucine and arginine. Catalytic measurements show a dramatic decrease of the activity in the chemically modified enzyme. Electrostatic potential calculations evidence in the modified enzyme a large contraction of the positive potential areas which surround the active sites in the native enzyme, indicating that electrostatic factors are critical in the enzyme-substrate encounter process of Mn- and Fe-superoxide dismutases. The activity drastically decreases also in Lys175-->Ile but not in the Lys175-->Arg mutant. Brownian dynamics simulations indicate that the decrease of activity in the Lys175-->Ile mutant cannot be due only to a decrease of the enzyme-substrate association rate, suggesting that Lys175 plays a relevant role also in the structural stabilization of the active site.
Gabbianelli
R
R
Department of Biology, University of Rome Tor Vergata, Italy.
Battistoni
A
A
Polticelli
F
F
Meier
B
B
Schmidt
M
M
Rotilio
G
G
Desideri
A
A
D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011424:Propionibacterium / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D013482:Superoxide Dismutase; D008239:Lysine